The overall objective of this proposal is to study the regulation of the HIV-1 life cycle in human astrocytes in vitro and the effects of this infection on astrocyte functions relevant to neuropathogenesis. Astrocytes can be infected with HIV-1 in vitro and in vivo but, unlike lymphocytes, they restrict HIV-1 replication. Analysis of HIV life cycle in astrocytes has been limited by inefficient infection of these cells. We have developed a new model system that permits initial high-level infection of human fetal astrocytes with HIV-1 by exposure of the cells to virus pseudotyped with MLV or VSV envelopes. High-level virus expression in astrocytes is followed by severe down-modulation of virus. We hypothesize that the efficient early HIV-1 expression in astrocytes induces cellular restrictions to further viral replication and alters the infected cell physiology. We will test this hypothesis by investigating the distinct phases of HIV-1 life cycle revealed in our system, including the early phase of high level viral replication and the late, chronic low-productive/inducible phase, and by studying selected cell functions affected by this infection. Our Specific Aims are: 1) To identify elements of HIV-1 replication in astrocytes that may contribute to neuropathogenesis in the brain; 2) To determine if the shift from productive to restricted HIV-1 infection in astrocytes is related to a changes in the activities of Tat or Rev; 3) To determine whether the shift from productive to latent HIV-1 infection in astrocytes is related to a change in selected cellular transcriptional factors acting on the HIV-1 LTR; 4) To investigate the effects of HIV-1 infection of astrocytes on activation of selected cellular genes and functions relevant to neurodegeneration; 5) To determine whether HIV-1 infection of astrocytes induces apoptosis or necrosis. The techniques employed will include cultures of human primary astrocytes; evaluation of viral expression by Southern, Northern, and Western blots, and PCR-based assays; assays of LTR-driven marker gene expression; measurement of chemokine and cytokine expression by Elisa; evaluation of cellular transcription factor expression by gel shift assays; and testing the induction of apoptosis-related genes and apoptosis. These studies will elucidate the molecular mechanisms governing the HIV-1 life cycle in astrocytes and aid the development of strategies for preventing their HIV-1 infection.